An efficient method for isolation of Hantaan virus through serial passages in suckling mice.

OBJECTIVE: Hantaan virus (HTNV) is one of the main etiologic agents for hemorrhagic fever with renal syndrome in China. However, it is very difficult to isolate the virus from its original host, which hampers the viral characterization. This study describes an efficient method for isolating HTNV in suckling mice. METHODS: Hantavirus-infected Apodemus agrarius were screened by quantitative real-time PCR. The homogenates of one positive rodent lung tissue were inoculated into suckling mice for virus propagation through serial passages. RESULTS: During the three passages in suckling mice, the number of viral RNA copies/nanogram of GAPDH mRNA increased significantly ranging from 477 to 7,278 and 46 to 4,898 in the tissues of brain and lung, respectively. Hantaviral antigens could be detected by indirect immunofluorescence assay and around 100-nm virion-like structures were also observed in brain tissue by transmission electron microscopy. No nucleotide exchange was found except for one in the 3'-non-coding domain of S segment when comparing the complete genome sequences from hantavirus in the first and the third passages. CONCLUSION: These results suggest inoculation of suckling mice with suspected hantavirus-infected rodent samples is an efficient method for isolation and maintenance of HTNV.

Structural studies of Hantaan virus.

Hantaan virus is the prototypic member of the Hantavirus genus within the family Bunyaviridae and is a causative agent of the potentially fatal hemorrhagic fever with renal syndrome. The Bunyaviridae are a family of negative-sense RNA viruses with three-part segmented genomes. Virions are enveloped and decorated with spikes derived from a pair of glycoproteins (Gn and Gc). Here, we present cryo-electron tomography and single-particle cryo-electron microscopy studies of Hantaan virus virions. We have determined the structure of the tetrameric Gn-Gc spike complex to a resolution of 2.5 nm and show that spikes are ordered in lattices on the virion surface. Large cytoplasmic extensions associated with each Gn-Gc spike also form a lattice on the inner surface of the viral membrane. Rod-shaped ribonucleoprotein complexes are arranged into nearly parallel pairs and triplets within virions. Our results differ from the T=12 icosahedral organization found for some bunyaviruses. However, a comparison of our results with the previous tomographic studies of the nonpathogenic Tula hantavirus indicates a common structural organization for hantaviruses.

Ex vivo stability of the rodent-borne Hantaan virus in comparison to that of arthropod-borne members of the Bunyaviridae family.

The possible effect of virus adaptation to different transmission routes on virus stability in the environment is not well known. In this study we have compared the stabilities of three viruses within the Bunyaviridae family: the rodent-borne Hantavirus Hantaan virus (HTNV), the sand fly-borne Phlebovirus sandfly fever Sicilian virus (SFSV), and the tick-borne Nairovirus Crimean-Congo hemorrhagic fever virus (CCHFV). These viruses differ in their transmission routes: SFSV and CCHFV are vector borne, whereas HTNV is spread directly between its hosts, and to humans, via the environment. We studied whether these viruses differed regarding stability when kept outside of the host. Viral survival was analyzed at different time points upon exposure to different temperatures (4 degrees C, 20 degrees C, and 37 degrees C) and drying at 20 degrees C. We observed clearly different stabilities under wet conditions, particularly at 4 degrees C, where infectious SFSV, HTNV, and CCHFV were detectable after 528, 96, and 15 days, respectively. All three viruses were equally sensitive to drying, as shown by drying on aluminum discs. Furthermore, HTNV and SFSV partially survived for 2 min in 30% ethanol, whereas CCHFV did not. Electron microscopy images of HTNV, SSFSV, and CCHFV stored at 37 degrees C until infectivity was lost still showed the occurrence of virions, but with abnormal shapes and densities compared to those of the nonincubated samples. In conclusion, our study points out important differences in ex vivo stability among viruses within the Bunyaviridae family.

Transmission electron microscopic study of the hemorrhagic spots in patients with epidemic hemorrhagic fever in the early stage.

Epidemic hemorrhagic fever (EHF) virus particles were found in the squamous epithelial cells and the capillary endothelial cells of the petechial spots located on the mucous membrane of the soft palate in 3 patients with severe early-stage EHF with transmission electron microscopy. The virus particles were round or oval in shape, about 100 nm in diameter, with a two-layer lipid envelope from which spikes were protruding. The nucleocapsid of the virus appeared to be hollow microfilamentoid or dense granules. Meanwhile, budding virus particles with a diameter of 80 nm were found in the enlarged Golgi apparatus. The infected cells displayed an enlarged and proliferating Golgi apparatus. The morphological characteristics of the viron mentioned above coincided with those of the virus particles of the family Bunyaviridae. This study is the first to demonstrate that the squamous epithelial cells on the mucous membrane of the soft palate are the target cells of EHF infection and to provide subcellular morphological evidence of petechial hemorrhage at the soft palate.

[Electron microscopic study on the petechial hemorrhagic spots in patients with epidemic hemorrhage fever (EHF)].

EHF viral particles were found in the squamous epithelial cells and capillary endothelial cells of the petechial spots located at the mucous membrane of the soft palate in cases of early stage of severe type EHF by transmission electron microscopy. The viral particles are round or oval in shape, about 100 nm in diameter with a lipid bilayer envelope from which spikes are protruding. The virions matured by budding through the intracytoplasmic membranes into the smooth surfaced vesicles. The morphological characteristics of the virion coincided with the viral particles of Family Bunyaviridae. It was the first time to demonstrate that the squamous epithelial cells of the soft palate is one of the target cells in EHF virus infection and to describe the subcellular morphological evidence of the petechial spots at the soft palate by EM.

[Propagation of the HTV in primary human embryonic kidney and lung cell culture].

2 strains of Hantaan virus (HTV, 76-118, Hubei-114) have been propagated successfully in cultured primary human embryonic kidney (HEK) and lung (HEL) cells. Cytopathic effect (CPE) was observed in the two kind of cells on day 5 to 7 postinoculation which showed the cell became round and clustered, then detached. The replicating peak of the Hubei-114 in two kinds of cell cultures appeared on the 11th day and another strain on the 14th or 17th day after infection. The ultrastructure changes were observed with EM and IEM, which stained by ICGT before embedding. It was discovered that the mitochondia atrophied and decreased, and inclusion bodies in the cytoplasma of HEK and KEL cells. A large amount of gold granulae were found in the inclusion bodies and the virions were seen occasionally. Contamination with other agents have been ruled out. Our data suggest that the replicating characters of HTV in these cell systems might be possible for the pathogenicity of HFRS for human.